This invention relates to compounds and compositions which modulate the NMDA receptor and, more specifically, which modulate the receptor through a novel complex site.
The N-methyl-D-aspartate (NMDA) receptor is a postsynaptic, ionotropic receptor which is responsive to, inter alia, the excitatory amino acids glutamate and glycine and the synthetic compound NMDA, hence the receptor name. The NMDA receptor controls the flow of both divalent (Ca++) and monovalent (Na+, K+) ions into the postsynaptic neural cell through a receptor associated channel. Foster et al., xe2x80x9cTaking apart NMDA receptorsxe2x80x9d, Nature, 329:395-396, 1987; Mayer et al., xe2x80x9cExcitatory amino acid receptors, second messengers and regulation of intracellular Ca2+ in mammalian neurons,xe2x80x9d Trends in Pharmacol. Sci., 11:254-260, 1990.
The NMDA receptor has been implicated during development in specifying neuronal architecture and synaptic connectivity, and may be involved in experience dependent synaptic modifications. In addition, NMDA receptors are also thought to be involved in long term potentiation, Central Nervous System (CNS) plasticity, cognitive processes, memory acquisition, retention, and learning. Furthermore, the NMDA receptor has also drawn particular interest since it appears to be involved in a broad spectrum of CNS disorders.
For instance, during brain ischemia caused by stroke or traumatic injury, excessive amounts of the excitatory amino acid glutamate are released from damaged or oxygen deprived neurons. This excess glutamate binds to the NMDA receptor which opens the ligand-gated ion channel thereby allowing Ca++ influx producing a high level of intracellular Ca++ which activates biochemical cascades resulting in protein, DNA, and membrane degradation leading to cell death. This phenomenon, known as excitotoxicity, is also thought to be responsible for the neurological damage associated with other disorders ranging from hypoglycemia and cardiac arrest to epilepsy. In addition, there are preliminary reports indicating similar involvement in the chronic neurodegeneration of Huntington""s, Parkinson""s, and Alzheimer""s diseases. Activation of the NMDA receptor has been shown to be responsible for post-stroke convulsions, and, in certain models of epilepsy, activation of the NMDA receptor has been shown to be necessary for the generation of seizures.
Neuropsychiatric involvement of the NMDA receptor has also been recognized. Blockage of the NMDA receptor Ca++ channel by the animal anesthetic PCP (phencyclidine) produces a psychotic state in humans similar to schizophrenia (reviewed in Johnson et al., xe2x80x9cNeuropharmacology of Phencyclidine: Basic Mechanisms and Therapeutic Potential,xe2x80x9d Annu. Rev. Pharmacol. Toxicol., 30:707-750, 1990.) Further, NMDA receptors have also been implicated in certain types of spatial learning. Bliss et al., Nature, 361:31 (1993). Interestingly, both the spatial and temporal distribution of NMDA receptors in mammalian nervous systems have been found to vary. Thus, cells may produce NMDA receptors at different times in their life cycles and not all neural cells may utilize the NMDA receptor.
Due to its broad-spectrum of neurological involvement, yet non-universal distribution, investigators have been interested in the identification and development of drugs acting at the NMDA receptor. Drugs acting on the NMDA receptor are, therefore, expected to have enormous therapeutic potential. For instance, U.S. Pat. No. 4,904,681, issued to Cordi et al. (Cordi I), describes the use of D-Cycloserine, which was known to modulate the NMDA receptor, to improve/enhance memory and to treat cognitive deficits linked to a neurological disorder. D-Cycloserine is described as a glycine agonist which binds to the strychnine-insensitive glycine receptor.
U.S. Pat. No. 5,061,721, issued to Cordi et al. (Cordi II), describes the use of a combination of D-cycloserine and D-alanine to treat Alzheimer""s disease, age-associated memory impairment, learning deficits, and psychotic disorders, as well as to improve memory or learning in healthy individuals. D-alanine is administered in combination with D-Cycloserine to reduce the side effects observed in clinical trials of D-Cycloserine, mainly those due to its growth-inhibiting effect on bacteria resulting in depletion of natural intestinal flora. D-Alanine reverses the growth-inhibiting effect of D-Cycloserine on bacteria. It is also reported that D-Cycloserine actually has partial agonist character.
U.S. Pat. No. 5,086,072, issued to Trullas et al., describes the use of 1-aminocyclopropanecarboxylic acid (ACPC), which was known to modulate the NMDA receptor as a partial agonist of the strychnine-insensitive glycine binding site, to treat mood disorders including major depression, bipolar disorder, dysthymia and seasonal effective disorder. It is also therein described that ACPC mimics the actions of clinically effective antidepressants in animal models. Again, in the examples provided, the compound was administered ip. In addition, a copending U.S. patent application is cited that describes that ACPC and its derivatives may be used to treat neuropharmacological disorders resulting from excessive activation of the NMDA receptor.
None of the foregoing offers, however, a satisfactory mechanism for modulating NMDA receptor function. Since glycine is necessary for receptor function, compounds modulating the glycine site offer a limited range of control. Further, glycine displays only limited sub-type specificity and compounds modulating the glycine site are expected to behave similarly.
Development of drugs targeting the NMDA receptor, although desirous, has been hindered because the structure of the NMDA receptor has not yet been completely elucidated. It is believed to consist of several protein chains (subunits) embedded in the postsynaptic membrane. The first two subunits determined so far form a large extracellular region which probably contains most of the allosteric binding sites, several transmembrane regions looped and folded to form a pore or channel which is permeable to Ca++, and a carboxyl terminal region with an as yet unknown function. The opening and closing of the channel is regulated by the binding of various ligands to domains of the protein residing on the extracellular surface and separate from the channel. As such, these ligands are all known as allosteric ligands. The binding of two co-agonist ligandsxe2x80x94glycine and glutamatexe2x80x94is thought to effect a conformational change in the overall structure of the protein which is ultimately reflected in the channel opening, partially opening, partially closing, or closing. The binding of other allosteric ligands modulates the conformational change caused or effected by glutamate and glycine.
A representation of the NMDA receptor showing schematically the principal recognition/binding sites which had been elucidated in the literature is depicted in FIG. 1. The sites marked xe2x80x9cGluxe2x80x9d and xe2x80x9cGlyxe2x80x9d are the receptor sites for the principal excitatory amino acid neurotransmitters, glutamate and glycine. The glutamate site also selectively binds NMDA. Since the binding of glutamate and glycine has been shown to stimulate the flow of Ca++ through the channel, glutamate and glycine are said to have a co-agonist (stimulatory) activity. Several competitive inhibitors of the actions of glutamate or glycine also bind to these sites and include those identified in the boxes in FIG. 1 labeled xe2x80x9cNMDA Antagonistsxe2x80x9d and xe2x80x9cGlycine Antagonists.xe2x80x9d Since these competitive inhibitors of the glutamate site block the flow of Ca++ through the channel, they are said to have an antagonist activity. The ligand-gated ion channel of the NMDA receptor is, thus, under the control of at least two distinct allosteric sites.
Two subunits of the mouse NMDA receptor channel have been identified by cloning and expression of complementary DNAs designated NR1 and NR2. Four subtypes of NR2 have been identified: NR2a, NR2b, NR2c, and NR2d. The heteromeric NR1/NR2a, NR1/NR2b and NR1/NR2c NMDA receptor channels exhibit distinct functional properties in affinities for agonists and sensitivities to competitive antagonists and Mg2+, block. In contrast to the wide distribution of the NR1 and NR2a subunit messenger RNAs in the brain, the NR2b subunit mRNA is expressed only in the forebrain and the NR2c subunit mRNA is found predominantly in the cerebellum. These findings suggest that the molecular diversity of the NR2 subunit family underlies the functional heterogeneity of the NMDA receptor channel. Kutsuwada et al, Nature, 358:36-40 (1992).
Several compounds are known which are antagonistic to the flow of cations through the NMDA receptor but which do not competitively inhibit the binding of allosteric ligands to any of the known sites. Instead, these compounds bind inside the open cation channel and are generally known as channel blockers. These are shown in FIG. 1 in the box labeled xe2x80x9cChannel Blockers.xe2x80x9d In fact, binding of a radiolabeled form of one such channel blocker, dizocilpine (i.e., [3H]MK-801), is a good measure of the activation of the NMDA receptor complex. When the channel is open, [3H]MK-801 may freely pass into the channel and bind to its recognition site in the channel. Conversely, when the channel is closed, [3H]MK-801 may not freely pass into the channel and bind. When the channel is partially open (partially closed) less [3H]MK-801 is able to bind than when the channel is fully open.
Channel blockers such as MK-801 and antagonists are known to protect cells from excitotoxic death, but in their case the cure may be as undesirable as the death since they block any flux of Ca++ thereby eliminating any chance of resumed normal activity. Channel blockers and glutamate site antagonists are known to cause hallucinations, high blood pressure, loss of coordination, vacuolation in the brain, learning disability and memory loss. PCP, discussed previously, produces a schizophrenic state in man.
Mg++ and Zn++ also modulate the NMDA receptor. The exact location of the divalent cation binding sites is still unclear. Zn++ appears to be antagonistic to channel opening and appears to bind to an extracellular domain. Mg++ shows a biphasic activation curvexe2x80x94at low concentrations, it is an agonist for NMDA receptor function and at high concentrations it is an antagonist. It appears to be absolutely necessary for proper receptor functioning and appears to bind at two sitesxe2x80x94a voltage dependant binding site for Mg++ within the channel and another non-voltage dependent binding site on the extracellular domain. These sites are also indicated in FIG. 1 by xe2x80x9cMg++xe2x80x9d and xe2x80x9cZn++xe2x80x9d.
It is believed that the channel is in constant motion, alternating between a cation passing (open) and a cation blocking (closed) state. It is not known at present whether the allosteric modulators actually increase the time during which the channel is open to the flow of ions, or whether the modulators increase the frequency of opening. Both effects might be occurring at the same time. Thus, the terms open and close, or agonistic and antagonistic, as used herein refer to a time averaged affect.
Recently, a third class of agonists which modulate the excitatory synaptic transmission at the NMDA receptor has been identified. (Ransom et al., xe2x80x9cCooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines,xe2x80x9d J. Neurochem., 51:830-836, 1988; Reynolds et al., xe2x80x9cIfenprodil is a novel type of N-methyl-D-aspartate receptor antagonist: interaction with polyaminesxe2x80x9d Molec. Pharmacol., 36:758-765, 1989; reviewed in Williams et al., xe2x80x9cModulation of the NMDA Receptor by Polyamines,xe2x80x9d Life Sci., 48:469-498, 1991.) These agonists are polyamines, principally the endogenous polyamines spermine and spermidine, which bind to other extracellular allosteric sites on the NMDA receptor. In FIG. 1, the allosteric polyamine binding site is labeled xe2x80x9cPA.xe2x80x9d The polyamines do not bind to the glutamate/NMDA site, the glycine site, or the channel blocker sites. However, polyamines do also bind inside the channel. There may be some relation between the Mg++ binding sites and the polyamine sites, but this relationship has not yet been fully elucidated. In contrast, there is strong evidence accumulating that polyamine binding is not thought to be necessary for functioning/activation of the NMDA receptor coupled cation channel, but is necessary for maximum activation. The polyamines, thus, are allosteric modulators of the NMDA receptor.
There appears to be a broad range of polyamine (diamine, triamine, and tetraamine) compounds which will modulate the site, some of which appear to be agonists, others partial agonists, and, finally, some antagonists. Binding of the compound 1,10-diaminodecane (DA10) decreases rather than increases the channel opening. This activity has been termed xe2x80x9cinverse agonistxe2x80x9d activity. Such inverse agonist binds competitively at the same site as an agonist but produces the opposite effect as the agonist.
Ideally, a drug for regulating the NMDA receptor will modulate the response of one of the endogenous ligands, and not itself be an endogenous ligand. The drug must be specific, i.e., it must affect an identifiable molecular mechanism unique to target cells that bear receptors for that drug.
It is therefore an object of the present invention to provide a class of specific drugs through the discovery of a novel binding site on the NMDA receptor for a compound which is not an endogenous ligand of the receptor.
It is a further object of the present invention to provide novel compounds for regulating the flow of Ca++ through the NMDA receptor, and compositions and methods for treating neurodegenerative disorders linked to NMDA receptor function.
Compounds, derived from the snail peptide Conantokin-G, act as allosteric modulators of the NMDA receptor cation channel and have effects ranging from inhibitory to partial modulatory to fully stimulatory on the polyamine or a closely associated modulatory site of the NMDA receptor. These compounds, therapeutic compositions, and their use for 1) treating neurological, neuropsychological, neuropsychiatric, neurodegenerative, neuropsychopharmacological and functional disorders associated with excessive or insufficient activation of the glutamate subtype of the NMDA receptor; 2) treating cognitive disorders associated with suboptimal activation or deactivation of the glutamate subtype of the NMDA receptor; and 3) improving and enhancing memory, learning, and associated mental processes, are disclosed. Examples of these disorders include acute or chronic neurodegenerative diseases, seizures, depression, anxiety, and substance addiction. The compositions can also be used to enhance learning and memory.
In one aspect, the compounds have the following formula (Formula I): 
wherein
A0 is an amino acid selected from the group consisting of natural, modified, or non-natural amino acids;
A1 is an uncharged, hydrophobic amino acid;
A2, A3 and A4 are amino acids independently selected from glutamate, aspartic acid, xcex3-carboxyglutamate (Gla), 3-carboxyaspartic acid, D-glutamate, phosphoserine, or phosphothreonine;
A5 is an uncharged, hydrophobic amino acid;
A6 is a peptide chain of from about 2 to about 15 amino acids, said amino acids selected from natural, modified, or non-natural amino acids;
A7 is an amino acid selected from the group consisting of natural, modified, or non-natural amino acids;
A8 is a basic amino acid selected from lysine or arginine;
A9 and A10 are amino acids selected from the group consisting of natural, modified, or non-natural amino acids;
R1 is H, Clxe2x80x94C6-COxe2x80x94, -benzoyl, or -benzoyloxy;
R2 is H or Clxe2x80x94C6-alkyl;
xa xb, xc, and xd are independently 0 or 1;
m and n are independently 0 or 1;
provided that m and n may not both be 0; and
pharmaceutically acceptable salts thereof.
It is provided that the compound of Formula I cannot be xa, is 0, A1 is glycine, A2 is glutamate, A3 is xcex3-carboxyglutamate, A4 is xcex3-carboxyglutamate, A5 is leucine, A6 is a peptide chain of 8 amino acids of the following composition Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg, A7 is xcex3-carboxyglutamate, A8 is lysine, A9 is serine, A10 is asparagine, and R1 and R2 are H.
It is further provided that the compound of Formula I is preferably not xa is 0, A1 is glycine, A2 is glutamate, A3 is glutamate, A4 is glutamate, A5 is leucine, A6 is a peptide chain of 8 amino acids of the following composition Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg, A7 is glutamate, A8 is lysine, A9 is serine, A10 is asparagine, and R1 and R2 are H.
Preferred compounds are compounds of Formula I where A6 is a peptide chain consisting of from about 7 to about 9 natural and/or non-natural amino acids;
R1 is H;
R2 is H; and
Xa is 0;
and pharmaceutically acceptable salts thereof.
Further preferred compounds of formula I wherein A1 and A5 are amino acids selected from the group glycine, alanine, valine, leucine, or isoleucine.
More preferred compounds ate compounds of Formula I wherein: A6 is a peptide chain consisting of about 8 natural amino acids.
Compounds preferred for their antagonistic properties are compounds of Formula I wherein n is zero.
Compounds preferred for their agonistic properties are compounds of Formula I wherein m is zero.
Specifically preferred for their modulatory activity are the following compounds:
Gly-Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:5);
Tyr-Gly-Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:6);
Ile-Arg-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:7);
Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:10);
Gly-D-Glu-D-Glu-D-Glu-Leu-Gln-D-Glu-Asn-Gln-D-Glu-Leu-Ile-Arg-D-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:11);
Gly-Glu-Ala-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:22);
Gly-Glu-Ser-GLa-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO: 23);
Gly-Glu-Ser(p)-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:24);
AcTyr-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-eu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:13);
Asn-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:14);
Asn(GlcNAc)-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:15);
Phe-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:16);
tBuTyr-Gly-Glu-Gla-Gla-Ieu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:20);
Ser-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:21);
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ala-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:35);
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ser-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:36);
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ser(p)-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:37);
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Ala-Lys-Ser-Asn-NH2 (SEQ ID NO:38);
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Ser-Lys-Ser-Asn-NH2 (SEQ ID NO: 39);
Gly-Glu-Gla-Gla-Leu-Gln-Glu-Asn-Gin-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH2 (SEQ ID NO:41);
Gly-Glu-Gla-Gla-Leu-Gln-Ala-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:32);
Gly-Glu-Gla-Gla-Leu-Gln-Ala-Asn-Gin-Ala-Leu-Ile-Arg-Ala-Lys-Ser-Asn-NH2 (SEQ ID NO:42);
Gly-Glu-Gla-Gla-Leu-Gln-Ser-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO: 33);
Gly-Glu-Gla-Gla-Leu-Gln-Ser(p)-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:34);
Gly-Glu-Gla-Gla-Leu-Gln-iodoTyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:53);
Gly-Glu-Gla-Gla-Leu-Gln-di-iodoTyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:54);
Gly-Glu-Gla-Gla-Leu-Gln-Tyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:31);
Gly-Glu-Gla-Gla-Leu-NH2 (SEQ ID NO:52);
Gla-Lys-Ser-Asn-NH2 (SEQ ID NO:49);
Ile-Arg-Gla-Asn-NH2 (SEQ ID NO:50); and
Lys-Ser-Asn-NH2 (SEQ ID NO:51).